Chimeric Ligands of Pili and Lectin A Inhibit Tolerance, Persistence, and Virulence Factors of Pseudomonas aeruginosa over a Wide Range of Phenotypes

Bacteria readily form resilient phenotypes to counter environmental and antibiotic stresses. Here, we demonstrate a class of small molecules that inhibit a wide range of Pseudomonas aeruginosa phenotypes and enable antibiotics to kill previously tolerant bacteria, preventing the transition of tolerant bacteria into a persistent population. We identified two proteins, type IV pili and lectin LecA, as receptors for our molecules by methods including a new label-free assay based on bacterial motility sensing the chemicals in the environment, the chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure–activity relationship studies reveal a molecule that inhibits only pili appendage and a class of chimeric ligands that inhibit both LecA and pili. Important structural elements of the ligand are identified for each protein. This selective ligand binding identifies the phenotypes each protein receptor controls. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and is correlated with killing previously tolerant bacteria. Inhibiting pili appendages impedes swarming and twitching motilities and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously controls the multiple signaling mechanisms preventing bacteria to elude antibiotic treatments.


Supporting Figures
Pellicle formation in wt PAO1 were observed and quantified by Congo red (CR) binding assay. Pellicles were formed for 3 days under static conditions. Overnight wt PAO1 bacterial cultures were diluted 1:100 in LB medium without NaCl with and without 85 µM 3,5-diMeDβM (βM) or 3,5-diMeDβC (βC) in the presence of 0.3 µg/ml tobramycin (0. . The cultures were grown statically (in glass tubes) for 3 days at 37°C. Then Congo red binding by pellicles was calculated as the difference between absorbance for reference solution containing the starting concentration of Congo red (40 µg/ml) and the absorbance of the supernatant. Error bars indicate standard deviations of independent triplicates. The pellicles formed without tobramycin (0-Tob) was considered as control. *P = 0.02 vs control; **P <0.005 vs control as evaluated using two-tailed unpaired Student's t-test

S10
Round I Round II Figure S10. Externally added pilin protein and 3,5-diMeDβM in gel neutralize each other's swarming inhibition activities. Repetitions of pili-ligand binding assay. 100 µL of pili protein (1mg/ml) expressed from (PA1244N3-pAC46) was spread on the surface of 0.5% agar (10 cm in diameter) containing different concentrations (0-30 µM) of 3,5-diMeDβM, followed by bacterial inoculation (3 µL of culture, OD 600 = 0.6). Pictures were taken after the plates were incubated at 37 ℃ for 12 h and then room temperature for another 12 h. The swarming area was estimated by measuring diameter of swarm area on agar gel.  PAO1k (6x10 7 CFU/ml) pretreated with and without 60 µM SFEG 4 OH and infected with ~10 6 PFU/ml phage, 10 min, 100 rpm. The phages left in supernatant (not adsorbed on pili) were quantified by plaque formation assay. The % phage adsorption was calculated as 1-(titer in supernatant/ titer of phage added) ×100. 4 Error bars indicate standard deviations of independent triplicates. *P=0.01 vs control as evaluated using two-tailed unpaired Student's t-test.   25 The positively charged methylene blue form noncovalent complex preferentially with negatively charged surfactants and does not bind to nonionic surfactants. The methylene blue bound with negatively charged surfactants has an increased partition into the organic chloroform phase bought in contact with the aqueous solution. The methylene blue in the chloroform phase were measured for its UV absorbance, which infers the concentration of rhamnolipids (µg/ml) in the culture.

Bacterial strains and bacteriophage used in paper:
Freezer stocks of all strains (Supplementary for Bacterial strains) were stored at -80 o C in lysogeny broth (LB) with ~20% glycerol. All strains were grown in LB (10 g/l tryptone, 5 g/l yeast extract, and 10 g/l NaCl) or Mueller-Hinton Broth MHB (beef infusion solids, 2.0 g/l. casein hydrolysate, 17.5 g/l. starch, 1.5 g/l) at 37 o C with shaking at 250 rpm. All biofilm inhibition, dispersion assays were performed in LB or MHB medium, and plates were incubated at 37 o C under static conditions.
Blood agar for revealing small colony variants: To prepare the blood agar gel containing our agents for visualizing small colony variants (SCVs), 1.75 g of premixed powder (Thermo Scientific™ Oxoid™ Columbia Blood Agar Base) containing peptone mixture 25.1 g/l, soluble starch 1.0 g/l, sodium chloride 5.0 g/l, agar 12.0 g/l was mixed and autoclaved with 100 mL of sterile water. The solution was cooled briefly without solidifying and followed by adding 5 mL of sheep blood (Hemostat) and mixed gently by swirling. Portions of 20 mL of this solution was poured into a 50 mL falcon tube, followed by adding 170 µL of a stock solution of agents 3,5-diMeDβM/βC (10 mM) to obtain gel solutions containing 85 µM of 3,5-diMeDβM/βC. The 20 mL gel solutions were poured into petri dishes (10-cm diameter) and solidified for inoculation and visualizing colonies of SCVs.
Crystal violet dye-based biofilm assay 10 An overnight culture (100 µL) of bacteria in LB was diluted in 10 mL of M63 and incubated to reach an OD 600 value of ~0.1. The bacterial culture (150 µL) was added to the wells of MBEC™ microtiter plate, followed by predetermined volumes of 3,5-DiMeDβM/βC stock solution for targeted concentrations (6 wells/concentration). The MBEC™ plates were incubated under stationary conditions at 37 ℃ for 24 h. After incubation, the pegs were transferred and immersed into 96 wells containing sterile water (200 µL) twice to briefly rinse the biofilms to remove unattached or loosely attached bacteria and were dried at 37 ℃ for 30 min. The peg-attached biofilms were immersed into 96 wells containing crystal violet (CV) dye solution (150 µL, 0.1%) at ambient temperature for 30 min, to stain the biofilms. The CV-stained pegs were then washed twice with sterile water (200 µL). To solubilize the CR stains, 150 µL of 30% acetic acid solution was added to wells, and the pegs was immersed in the wells, the plates were shake on a microplate mixer (Scilogex MX-M) at 100 rpm for 15 min. The amount of biofilms was inferred and quantified by measuring the OD 600 of the 150 µL acetic acid solution on plate reader. The absorption from stained pegs containing just M63 medium was subtracted from pegs treated with agents.

Confocal microscopy of biofilms and image acquisition 1, 11
In a 24-well microtiter plate, biofilms were grown by placing 100 µL of wt PAO1 culture with and without tobramycin, and with and without 85µM 3,5-diMeDβM/βC, on polystyrene coupons (roughly 3/8 in. × 3/8 in.) that were cut from a polystyrene petri dish. The Saran-wrapped plate was then incubated at 37 o C without shaking. Each polystyrene coupon was then washed gently by immerging into saline twice and then placed on a microscope cover glass (50 x 24 mm, No. 2, Fisher Scientific, Pittsburgh, PA). All microscopy images were acquired using a Zeiss LSM confocal laser scanning microscope (Carl Zeiss, Germany) for monitoring Green Fluorescent protein (GFP) and propidium iodide (PI) fluorescence.
Pellicle formation and quantification 14 Pellicle formation assay was adopted as previously described. Briefly, overnight culture (100 µL) of wt PAO1 were inoculated in borosilicate glass tubes (18 mm by 150 mm) containing 10 mL of MHB supplemented with and without 0.3 µg/ml tobramycin, 85 µM 3,5-diMeDβM/βC. Tubes were incubated without shaking at 37°C for 3 days. Pellicle formed at the air-liquid interface was resuspended in 1.5 mL PBS buffer. Congo red stock solution (10 mg/ml) was added to a final concentration of 40 µg/ml and incubated with shaking for 2 h at 37°C. Following this incubation, the bacteria were pelleted via centrifugation (6000 rpm, 15 min); and the absorbance (490 nm) of 200 uL of the supernatant was measured on plate reader. The PBS with 40 µg/ml Congo red was used as the reference to determine the amount of Congo red bound to pellicles.
Quantification of biofilm bacteria and mass 1, 11 We quantified the killing of bacteria in biofilms under different chemical treatment (tobramycin and 3,5-diMeDβM/βC) by CFU counting, and measured biofilm mass by CV dye assay. An overnight culture (100 µL) of bacteria in LB was diluted in 10 mL of M63 and subcultured to reach an OD 600 value of ~0.1. The bacterial culture (150 µL) was added to the wells of MBEC™ microtiter plate, followed by predetermined volumes of 3,5-diMeDβM/βC stock solution for targeted concentrations (6 wells/concentration). The 24 hold biofilm on pegs were transferred to wells of saline briefly, and then transferred to wells containing 150 µL of fresh M63 medium containing of tobramycin, 3,5-diMeDβM or 3,5-diMeDβC with targeted concentrations and were incubated for an additional 0-24 h at 37 o C. The pegs were washed with saline and clipped with sterile plier. Each condition consisting of 6 pegs was sonicated in 5 ml of saline and serially diluted in saline followed by plating on LB agar plates for 24 hours for counting colony forming units. The amount of the biofilms on pegs were measured by using the crystal violet staining method described in biofilm inhibition assay. The bacterial count (CFU/ml) was normalized by amount of biofilm.

Hemolysis assay 15
Single donor Human Red Blood Cells (Innovotech) suspension (300 µL) was mixed with different volumes of 3,5-diMeDβM/C to reach final concentrations of 0-500 µM in PBS buffer, respectively. mix 300 µL of RBC suspensions with of 30 µL of Triton X (1%) and PBS, respectively as Positive and negative controls After incubation at 37 with 100 rpm shaking for 1 hthe samples were then centrifuged for 2 min at 2000 ℃ rpm. The absorbance of 200 µL supernatant was recorded at 540 nm. % Hemolysis was calculated as (OD sample − OD negative control )/(OD positive control − OD negative control ) × 100%. Experiments were performed in triplicate. 16 A 3,5-diMeDβM/βC stock solution (10 mM) was diluted in PBS buffer at various concentrations (0-200 µM). An ethanol solution (1 μL) of Nile red (2.5 mM) was added to 1 mL assay solution, and the fluorescence emission was measured on a using Synergy 2 multimode microplate reader an excitation S18 wavelength of 550 nm after 20 minutes of incubation at 37 . Fluorescence intensity was recorded at 635 ℃ nm. Experiments were performed in triplicate.

Twitching assays 18-19
Subsurface twitching assays were modified from previously reported procedures. Briefly, 100 µL of overnight culture of PAO1-EGFP was diluted in 900 μL of Luria Bertani broth without NaCl (LBNS). Sterile pipette tips (10 µL) were dipped in this LBNS suspension and were then stabbed through a one-dayold 1% LBNS agar, with and without 85 µM of 3,5-diMeDβM or 3,5-diMeDβC, to inoculate bacteria at bottom of the agar gel. Plates were incubated upright (not inverted) in an incubator at 37 ℃ for 5 days, and images of the plates were taken under ultraviolet light (Accuris™ E3000 UV Transilluminator -302nm) in dark room.

Gel electrophoresis of sheared surface proteins from wild type P. aeruginosa 20
The levels of pili on bacterial surface were analyzed by gel electrophoresis as described previously with slight modifications 20 . The wt PAO1 and transposon mutant pilT::Tn were streaked on LB agar plates with and without 85µM 3,5-diMeDβM/βC and incubated at 37 °C for ~ 16 h. The bacterial cells were scraped from the agar gels with glass coverslips and resuspended in 5 mL of phosphate buffered saline, pH 7.0, with and without 85µM 3,5-diMeDβM/βC, respectively. The solutions were centrifuged at 6000 rpm for 15 min to collect bacterial cells. The collected bacterial pellet was resuspended in 1 ml PBS buffer, and vortexed for 30 s to shear surface proteins from suspended bacterial cells. The suspensions were transferred to three separate 1.5 ml Eppendorf tubes and centrifuged at 11,688 x g for 5 min to pellet the bacterial cells. The supernatant was transferred to fresh tubes and centrifuged at 11,688 x g for 20 min to further pellet remaining cells. The supernatants were collected in new tubes, mixed with 1:10 volume of 5 M NaCl and 30% (w/v) polyethylene glycol (PEG 8000; Sigma-Aldrich), and incubated on ice for 90 min, to precipitate the surface proteins. The solutions were centrifuged at 11,688 x g for 20 min, to collect the aggregated proteins. The protein pellets were resuspended in 100 μL PBS buffer and the amount of total surface sheared protein was determined by Bradford assay that resulted for 120 µg/ml protein mass for wt PAO1 without 3,5-diMeDβM/βC treatment, and 136 µg/ml for wt PAO1 with 3,5-diMeDβM/C treatment, which were diluted to 120 µg/ml; and 470 µg/ml for transposon mutant pilT::Tn without 3,5-diMeDβM/βC treatment, which was diluted to 321 µg/ml, and 321 µg/ml for transposon mutant pilT with 3,5-diMeDβM/βC treatment. The protein samples (80 µL) were mixed with 150 μL of 1X sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris, pH 6.8, 2% β-mercaptoethanol, 20% glycerol, 4% SDS and 0.001% bromophenol blue), and boiled for 10 min, cooled to room temperature; 20 µL of the sample were loaded to for separation on 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were visualized by staining with Coomassie brilliant blue. 4 All phage counts were quantified by phage titer using top overlay method to report the plaque forming units. 4 Top agar preparation. Mix 3 mL of warm top agar solution (7 g/l agar, 0.5 g/l sodium chloride, 10 g/l tryptone, 5 g/l yeast extract in sterile water) and 200 µL of LB culture of PAO1k (OD 0.6); spread this mixture solution on 1.5% LB agar plate (LB-Miller, 10 g/l tryptone, 5 g/l yeast extract, 10 g/L NaCl ,15 g/l agar, 10-cm in diameter). Cool for 15 min in biosafety level 2 hood to solidify the gel. Serial dilutions of supernatant of containing phage were prepared in LB-Mg 2+ . Nine or six drops of 10 µL of the diluted supernatant were placed on the top agar plates to observe plaques formation on bacterial lawn after 12 h. The number of plaques were counted and reported as plaque forming units per ml of sample.

Direct binding of fluorescent ligands to LecA 23
The fluorescent ligand (βGal-aryl-Dansyl) were dissolved in water to a final concentration of 3 mg/ml. 2 mg LecA (Sigma) was dissolved in 1 mL of 0.1M Tris-HCl pH 7.5 supplemented with CaCl 2 (6 µM). The solution of fluorescent ligand (βGal-aryl-Dansyl, 200 nM) is mixed with different concentrations of LecA solution (0-100 µM). After incubation for 1 h at room temperature in dark, fluorescence polarization was measured by using Edinburgh FLS9801 Spectrometer, with the samples being illuminated with vertically polarized light at 330 nm (for βGal-aryl-Dansyl). The vertical and horizontal fluorescence components of emission 570 nm were recorded, and the fluorescence polarization was calculated and data were analyzed using a four-parameter fit model.

Competitive binding of ligands to LecA 23
To a solution of LecA (final concentration: 20 μM) and fluorescent ligand (final concentrations of βGalaryl-Dansyl: 200 nM) in 0.1M Tris-HCl pH 7.5 supplemented with CaCl 2 (6 μM), serial dilutions (0.1 μM to 140 μM) of test compounds (SFβM/C, rhamnolipids, SFEG 4 OH and 3,5-diMeDβM/βC) were done. The mixture samples were incubated for 6 hours at room temperature. The fluorescence polarization was measured using Edinburgh FLS9801 Spectrometer. During measurement, the samples were illuminated with vertically polarized light at 330 nm (for βGal-aryl-Dansyl) and vertical and horizontal fluorescence components were measured, and the fluorescence polarization was calculated and data were fitted with four parameter variable slope model.

Elastase assay 18, 24
Elastase activity in P. aeruginosa culture was determined by an elastin Congo red (ECR) assay with modifications 18,24 . Briefly, 100 µL of bacteria from overnight cultures in MHB were diluted in fresh MHB to an OD 600 of 0.01 with and without 3,5-diMeDβM or 3,5-diMeDβC, SFEG 4 OH (final concentration: 85 µM). After culturing for 18 h at 37°C with shaking, culture supernatants were filtered (0.45-µm pore-size filter). Triplicates of culture filtrates (100 µL) were added to 1 mL of Tris buffer (0.1 M Tris, pH 7.2, 1 mM CaCl 2 ) containing 20 mg of Elastin-Congo red (Sigma Aldrich). The glass tubes were incubated for 6 h at 37°C with shaking at 100 rpm and were added with 0.1 ml of 0.12 M EDTA and placed on ice. Insoluble ECR was removed by centrifugation at 16,000 rpm and the OD 495 of the supernatant was measured. The elastase activity was reported as OD 495 (with agent) -OD 495 (without agent).

Rhamnolipid assay 25
Quantification of rhamnolipids in planktonic cultures of P. aeruginosa was determined by methylene bluerhamnolipid complex assays by an established assay. 25 In this assay, the positively charged methylene blue form noncovalent complex preferentially with negatively charged surfactants, and does not bind to nonionic surfactants. The methylene blue bound with negatively charged surfactants has an increased partition into the chloroform phase bought in contact with the aqueous solution. The methylene blue in the chloroform phase were measured for its UV absorbance, which infers the concentration of negatively surfactants. Briefly, 100 µL bacteria from overnight cultures in MHB were diluted in fresh MHB to an OD 600 of 0.01 with and without 3,5-diMeDβM or 3,5-diMeDβC, SFEG 4 OH (final concentration: 85 µM). After 18 h at 37°C with shaking, culture supernatants were filtered with 0.45-µm pore-size filter (SCBT, USA). The filtrate pH was first adjusted to 2.3 ± 0.2 using 1 N HCl. The acidified sample was then extracted with fivefold volume of chloroform. The chloroform extract (4 mL) was transferred to put in contact with 5 mL of a freshly prepared methylene blue aqueous solution (40 µg/ml). The pH of this aqueous solution of methylene blue is pre-adjusted to 8.6 ± 0.2 by adding the 50 mM borax buffer. After 4 mins vortex shaking, the samples were left to stand in dark for 15 min. The bottom chloroform phase (1 mL) was transferred into a cuvette and the absorbance was measured at 638 nm with a UV/Vis spectrophotometer with a reference of blank chloroform. The absorbance values were converted to rhamnolipid concentrations using a calibration curve.
Pyocyanin assay 26 An overnight culture of PAO1 (100 µL) was added to 10 mL fresh LB medium, 5 mL of which were grown with and without 85 µM 3,5-diMeDβM, or 3,5-diMeDβC, SFEG 4 OH for 12 h. The absorbance of bacterial culture was measured at 600 nm (A 600 ), and then centrifuged to remove bacterial pellet. Supernatant (5 mL) was extracted with 3 mL chloroform. The pyocyanin-containing chloroform layer was acidified with 2 mL of 0.1 N HCl to give a pink solution, the absorbance at 520 nm of 200 µL of the solution was measured. The absorbance reading at 520 nm was converted into µg/ml of supernatant with standard curve reported in literature. Pyocyanin levels were expressed as µg/ml of supernatant/A 600 of culture where A 600 is absorbance of bacterial culture.

2
To a solution of 1 (2.78 g, 22 mmol) in dry acetonitrile (6 mL) triethylamine (11.5 mL), trimethylchlorosilane (4.1 mL) and sodium iodide (4.63 g, 30.9 mmol) dissolved in acetonitrile (29 mL) were added successively. The reaction mixture was stirred at room temperature. After 4 h, cold water (37 mL) was added and the aqueous solution extracted with cold hexane. The organic layers were combined and evaporated in vacuo to give 2 as a brown oil. Crude product was assessed by NMR and deemed pure enough use crude in next step without further purification (81% yield). A solution of silver trifluoroacetate (2.09 g, 9.45 mmol) suspended in dichloromethane (9 mL) was cooled to -78 o C and Compound 2 (1.79 g, 9 mmol) was added dropwise. 1-iodohexane (2.0 g, 9.45 mmol) was then added and the reaction was allowed to warm to room temperature then after ten minutes the reaction mixture was filtered and concentrated en vacuo. The crude product was flash chromatographed on silica gel (hexane/AcOEt 95 : 5) to yield 3 as a colorless oil (19% yield A solution of 3 (160 mg, 0.76 mmol) in dry dichloromethane (2 mL) was cooled to 0ºC and mchloroperbenzoic acid (350 mg, 2.03 mmol) was added. The suspension and trifluoroacetic acid (0.288 mL, 3.76 mmol) was added dropwise. During this operation, the reaction flask was protected from light. Subsequently, the solution was allowed to warm to room temperature. After 16 h, the mixture was diluted with dichloromethane (2 mL) and the organic layer was washed successively with a 10% aqueous solution of Na 2 SO 3 (2 mL), a saturated aqueous solution of potassium carbonate (1 mL) and water (2 mL). Finally, the organic layer was evaporated in vacuo and the product purified by column chromatography (9:1 hexanes: ethyl acetate) to give lactone 4 (46% yield Under nitrogen atmosphere, a solution of 4 (1.608 g, 7.58 mmol) in a 10 mM solution in anhydrous methanol (7.1 mL) was stirred overnight at room temperature. Then, the reaction was neutralized using H + amberlite resins. After filtration, the filtrate was evaporated in vacuo to give the methyl ester 5 as a clear, oily liquid (87% yield A solution of 5 (1.098 g, 0.25 mmol) in dichloromethane (1.5 mL) was stirred on ice. Triethylamine (0.23 mL, 1.6 mmol) and mesyl chloride (655 mL, 0.51 mmol) were added. The mixture was stirred at 0ºC. After 4 h, aqueous NaHCO 3 (1N, 1.2 mL) was added and the resulting solution extracted with dichloromethane. The organic layers were combined and evaporated in vacuo and the product purified by column chromatography (9:1 hexanes: ethyl acetate)to give mesylate 6 as a yellowish oil (91% yield).  To a suspension of LAH (63 mg, 1.7 mmol) in anhydrous diethyl ether (1 mL) at 0ºC, a solution of 6 (68 mg, 0.20 mmol) in anhydrous ether (1 mL) was added. The mixture was brought to room temperature then refluxed for 16 h. Then, the solution was cooled to 0ºC before quenching with water. The resulting aqueous layer was extracted with ether. The combined organic fractions were evaporated in vacuo and the oily residue was flashchromatographed (hexane/AcOEt 9:1) to yield alcohol 7 as an oil (53% yield  Figure S16: Synthesis scheme for a fluroscent probe, βGal-aryl-Dansyl 4-Aminophenyl β-D-galactopyranoside (0.12 g, 1.2 mmol) in anhydrous DMF (2 mL) was added to Et 3 N (0.53 g, 0.53 mmol) in anhydrous DMF (5mL) at 0 ºC. To this solution was added dansyl chloride (0.41 g, 1.5 mmol). After stirring at 0 ºC for 2 hours, the mixture was concentrated, and the residue obtained was subjected to the purification by column chromatography DCM: